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SIRT4 Controls Macrophage Function and Wound Healing through Control of Protein Itaconylation in Mice

ABSTRACT: Proper regulation of inflammatory responses is essential for organismal health. Dysregulation can lead to accelerated development of the diseases of aging and the aging process itself. Here, we identify a novel enzymatic activity of the mitochondrial sirtuin SIRT4 as a lysine deitaconylase that regulates macrophage inflammatory responses. Itaconate is a metabolite abundantly produced in activated macrophages. We find it forms a protein modification called lysine itaconylation. Using biochemical and proteomics approaches, we demonstrate that SIRT4 efficiently removes this modification from target proteins both in vitro and in vivo. In macrophages, SIRT4 expression increases upon LPS stimulation, coinciding with elevated protein itaconylation. SIRT4-deficient macrophages exhibit significantly increased IL-1Î² production in response to LPS stimulation. This phenotype is intrinsic to macrophages, as demonstrated by both SIRT4 knockdown and lentiviral over-expression models. Mechanistically, we identify key enzymes in branched-chain amino acid (BCAA) metabolism as targets of hyperitaconylation in SIRT4-deficient macrophages. The BCKDH complex component dihydrolipoamide branched chain transacylase E2 (DBT) is hyperitaconylated in SIRT4KO macrophages, concomitant with reduced BCKDH activity. Physiologically, SIRT4-deficient mice exhibit significantly delayed wound healing, demonstrating a consequence of dysregulated macrophage function. Our data reveal a novel protein modification pathway in immune cells and establish SIRT4 as a critical regulator at the intersection of metabolism and inflammation. These findings have implications for understanding immune dysregulation in aging and metabolic disease.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Matthew D. Hirschey

PROVIDER: PXD063862 | JPOST Repository | Wed May 13 00:00:00 BST 2026

REPOSITORIES: jPOST

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			Action	DRS
	2019_01_18_Hirschey_SIRT4-BMMproteome_KApooledRun1-START_2019-0109_190130195710.raw	Raw
	2019_01_30_Hirschey_SIRT4-BMMproteome_KA13_2019-0109.raw	Raw
	2019_01_30_Hirschey_SIRT4-BMMproteome_KA14_2019-0109.raw	Raw
	2019_01_30_Hirschey_SIRT4-BMMproteome_KA15_2019-0109.raw	Raw
	2019_01_30_Hirschey_SIRT4-BMMproteome_KA16_2019-0109.raw	Raw

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Project description:Siglec-E is a murine CD33-related siglec that functions as an inhibitory receptor and is expressed mainly on neutrophils and macrophage populations. Recent studies have suggested that siglec-E is an important negative regulator of LPS-TLR4 signalling and one report (Wu et al 2016) claimed that siglec-E is required for TLR4 endocytosis following uptake of Escherichia coli by macrophages and dendritic cells (DCs). Our attempts to reproduce these observations using cells from wildtype (WT) and siglec E deficient mice were unsuccessful. We used a variety of assays to determine if siglec-E expressed by different macrophage populations can regulate TLR-4 signalling in response to LPS, but found no consistent differences in cytokine secretion in vitro and in vivo, comparing 3 different strains of siglec-E-deficient mice with matched WT controls. No evidence was found that the siglec-E deficiency was compensated by expression of siglecs-F and –G, the other murine inhibitory CD33-related siglecs. Quantitative proteomics was used as an unbiased approach and provided additional evidence that siglec-E does not suppress inflammatory TLR4 signaling. Interestingly, proteomics revealed a siglec E dependent alteration in macrophage phenotype that could be relevant to functional responses in host defence. In support of this, siglec-E-deficient mice exhibited enhanced growth of Salmonella enterica serovar Typhimurium in the liver following intravenous infection, but macrophages lacking siglec-E did not show altered uptake or killing of bacteria in vitro. Using various cell types including bone marrow derived DCs (BMDC), splenic DCs and macrophages from WT and siglec-E-deficient mice, we showed that siglec-E is not required for TLR4 endocytosis following E.coli uptake or LPS challenge. We failed to see expression of siglec-E by BMDC even after LPS-induced maturation, but confirmed previous studies that splenic DCs express low levels of siglec-E. Taken together, our findings do not support a major role of siglec-E in regulation of TLR4 signalling functions or TLR4 endocytosis in macrophages or DCs. Instead, they reveal that induction of siglec-E by LPS can modulate the phenotype of macrophages, the functional significance of which is currently unclear.

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SIRT4 Controls Macrophage Function and Wound Healing through Control of Protein Itaconylation in Mice

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